PROJECT SUMMARY Aging is associated with dysregulation of the immune response, which is also termed ?immunosenescence.? Each part of the immune system is influenced to some extent by the aging process. However, adaptive immunity seems more extensively affected, and it is especially the T cells that are altered. In fact, the number and proportion of late-differentiated T cells, particularly CD8+ T cells, is higher in the elderly than in the young and their accumulation may contribute to the enhanced systemic pro-inflammatory milieu commonly seen in elderly individuals. Interestingly, the proportion of CD8+ effector memory RA (TEMRA) cells increases significantly with age, which is not seen for CD4+ cells. We do not know exactly what causes these observed changes, but an understanding of the possible causes is now beginning to emerge. Spaceflight causes a suite of negative health effects that may be comparable to immunosenescence, which seems to be a key regulator of the regenerative capacity of tissue- and organ-specific stem cells. Organ-specific stem and progenitor cells may allow the design of strategies for organ regeneration. The overarching objective of this proposal is to gain a better understanding of the influence of immunosenescence on the regenerative capacity of tissue-specific stem cells. Specifically, studies examining the effects of bone healing (by mesenchymal stromal cells) and vascular regeneration (by endothelial progenitor cells) are planned using tissue mimics on chip to represent ?semi-3D? architectures. Ultimately, we will use microgravity as an aging model, and we will translate those findings to improve human health on Earth by using tissue chips on the ISS for up to a month. Notably, the proposed studies investigate post-flight recovery of tissue chips using histological analysis combined with functional and genomic analysis. In collaboration with Space Technology and Advanced Research Systems, Inc. - STaARS, well-versed in maintenance of cell culture experiments in spaceflight, we are establishing an in vitro tissue-on-chip platform that mimics human physiology to study the effect of immunosenescence on tissue-specific stem cells. This system will be designed for use in the UH3 phase in the extreme environment of space. In the UG3 phase, we will examine the effect of simulated microgravity and normal (1xg) conditions on in vitro cultures of CD8+ T cells and on their co-culture with stem cells (UG3 Aim 1). In UG3 Aim 2 we will investigate the effect of microgravity at the International Space Station ? National Lab (ISS-NL) on in vitro co-cultures of CD8+ T cells and stem cells. In the UH3 phase, we will determine the effect of microgravity on immunosenescence (differentiation of CD8+ T cells into TEMRA cells) and tissue-specific stem cells in space as model for aging using microarrays (UH3 Aim 1). Finally, in UH3 Aim 2, we will investigate post-flight recovery of tissue chips (from Aim 1, UH3 phase) using functional analysis of stem cells. This proposal will contribute materially to our understanding of the impact of an aged immune system on tissue healing and regeneration.